Functional Research Of Lysine Crotonylation Relating To Pluripotency Regulation In MESC

Background and purpose:Pluripotent stem cells(PSCs)can be propagated in vitro under different culture conditions,resulting in differentiated cell states with distinct properties.To understand how PSCs switch from one state to another,ultimately leading to lineage-specific differentiation,is important for developmental biology and regenerative medicine.Although there is much information regarding gene expression changes controlling these transitions,less is known about post-translational modifications of proteins on pluripotency regulation.Protein crotonylation is a newly discovered post-translational modifications attached to lysine residues by the metabolite crotonyl-CoA through crotonyltransferase.In our project,we want to explore the discrepancy and change of lysine crotonylated proteome in different PSCs and the potential impacts of lysine crotonylation on pluripotency regulation,however,this paper will mainly narrates and talk about the association of lysine crotonylation with pluripotency regulation in m ESCs cultured under serum/Lif condition.Methods:mESCs and mEpiSCs were derived from ICM of blastocyst prior to implantation and epiblast following implantation respectively and cultured in corresponding medium.Moreover,mESCs were cultured in three different medium including 2i/L,S/L and differentiated conditions.Subsequently,we collected PSCs under the four cultured conditions and combined affinity purification of crotonylated peptides and liquid chromatography-tandem mass spectrometry(LC-MS/MS)to systematically profile protein crotonylation in mouse PSCs in different states including na?ve,metastable,and metastable states undergoing early pluripotency exit of mESCs,as well as primed mEpiSCs.Results:We successfully identified 8,102 crotonylation sites distributing in 2,578 proteins,among which 1,426 proteins were with high-confidence.These high-confident crotonylated proteins are centered on factors concerning functions related to pluripotency regulation such as RNA biogenesis,central carbon metabolism,and proteasome mediated proteine quilibrium.Interestingly,we found that increasing the cellular level of crotonyl-CoA through crotonic acid treatment heightens proteasomeactivity in metastable ESCs cultured under S/L condtion and delays their differentiation.Moreover,we identified and validated some novel crotonylated proteins covering pluripotency regulator(LIN28A),RNA m6 A reader(YTHDF2)and hexokinase2(HK2).Conclusion:mESCs treated by crotonic acid can maintain pluripotency under normal S/L cultured condition and delay differentiation under differentiated condition,which indicates that lysine crotonylation is beneficial for the pluripotency maintenance of mESCs.As for the augmented activity of proteasome in mESCs treated by crotonic acid,it's reported that UPS plays an crutial role in the pluripotency regulation of PSCs,thus,we inferred that crotonic acid may promote the pluripotency preservation of mESCs though inhanced proteasomal activity.Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation including the role of metabolism in other cell fate transitions.